It is hypothesized that the specific cooperative interactions operative throughout the three-dimensional structure evolve in the late phase of folding to stabilize structure and that the mechanism of this global cooperative interactions, also possible related to the function of cytochrome c, would have been conserved throughout evolution for 1.5 billion years. Thus, testing this hypothesis by synthesis of the ancestor cytochrome c would be an effective approach to the study of the relationship between the amino acid sequence (translated from the genetic information) and the structure-function of proteins. Yeast mitochondrial cytochrome c synthetase which catalyzes the covalent attachment of the heme group to apaocytochrome c to form cytochrome c has now been solubilized using Triton X-100. The product formed by the action of the solubilized enzyme from hemin and apoprotein in the presence of NADPH was isolated. The isolated product exhibited Soret absorbance bands identical with those of cytochrome c, indicating the presence of two thio ether bonds. It is also found that the enzyme recognizes the chemically synmthesized fragment containing residues 1 to 25 as a substrate. Thus, in principle, the way for total synthesis of the ancestor cytochrome c is now opened.